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Enumeration, Isolation and Identification of Nitrogen-fixing Bacterial Strains at Seedling Stage in Rhizosphere of Rice Grown in Non-calcareous Grey Flood Plain Soil of Bangladesh

Md. Harunor Rashid KHAN
Department of Soil, Water and Environment, University of Dhaka, Dhaka 1000, Bangladesh.

Md. MOHIUDDIN
Department of Soil Science, Govt. Barisal College, Barisal, Bangladesh.

M. RAHMAN
Department of Soil, Water and Environment, University of Dhaka, Dhaka 1000, Bangladesh.

Abstract/ Executive Summary:

Non-symbiotic diazotrophic systems for biological nitrogen fixation (BNF) in agriculture are most promising but the possibility for the extension of nitrogen fixation by rice is still speculative. Accordingly, the present study was conducted for the Enumeration, isolation and identification of nitrogen fixing bacterial strains at seedling stage (30 days after seed sowing) in rhizosphere of rice (BR 10, Oryza sativa L.) grown in Non-Calcareous Grey Flood Plain soil of Bangladesh. The soil is classified as ‘Inceptisol’ order and ‘Aquept’ suborder. It was identified as ‘Dhamrai series’, had ‘silt’ texture, pH 7.1 and 5.5 C/N ratio. The present results of the microbial tests on the rice rhizosphere soil evinced that out of 263 isolates, only 91 were branded as nitrogen fixing organisms per gram of soil, which was about 34.6 % of the total isolates. As per selection criteria, four individual strains were considered for identification. Biochemical tests were conducted for proper identification and the selected strains were identified as Enterobacter spp., Klebsiella spp., Bacillus spp. and Azospirllum spp.

Keywords: Azospirllum spp., Bacillus spp., diazotrophs, Enterobacter Spp., Klebsiella spp., Oryza sativa

Location:

Start Date:

End Date:

Program Area: Resource Development and Management

Commodity: Micro organism, Rice

Objectives:

The aim of the present research was to characterize and identify nitrogen-fixing organisms at the seedling stage of rice rhizosphere soil in order to reduce the use of chemical nitrogen fertilizers by the production of biofertilizer.

Methodology:

2.1 Soil Sampling Soil at a depth of 0 to 15 cm was sampled during October from Dautia Bil at Dhamrai under the district of Dhaka at the seedling stage (30 days after seed sowing) of rice (BR 10, Oryza sativa L.). Six samples of rhizosphere soil were collected from an area of 100 m2. According to 1981 reviewed Reconnaissance Soil Survey report of Dhaka district, the studied site is identified as Non-Calcareous Grey Flood Plain soil as per the general soil type of Bangladesh and the collected soil is representing Dhamrai series. The soil responded well when Aus and Boro-aman were cultivated. The field was used to receive N-fertilizers at the rate of 60 kg ha^-1yr.^-1. In the winter season Rabi crops are cultivated. The soil was deeply flooded (about 1.5 to 2.5 m) during rainy season. 2.2 Dilution of Soil Samples The sampled rhizosphere soil was mixed thoroughly to make a composite soil. Then 10 gm of sub-soil sample diluted to 100 ml that considered being 10^-1 dilution factor. Transferring of 1 ml of 10^-1 dilution to 9 ml sterilized water with the help of a sterilized pipettes yielded 10^-2 dilution. In this way, a series of up to 10^-8dilution was prepared under aseptic condition. Screw cap test tubes and glass petridishes were used to culture microorganisms. Sterility is the hallmark for successful works in the microbiological studies were also kept in mind throughout the study. 2.3 Bacterial counts The calculation for the total numbers of bacteria was done by plating soil dilutions on nutrient agar and total numbers of N2 fixing bacteria were counted by plating soil dilutions on nitrogen free medium –RCV media. One ml of the suspension from each (10^-1, 10^-2, 10^-3, 10^-4, 10^-5, 10^-6, 10^-7, 10^-8) was taken and poured into the nutrient agar media and the nitrogen free media on petridish separately. Then incubated the plates at 30 ⁰C for 48 hours. The total count of the microorganisms was obtained by multiplying the number of cells per plate by the dilution factor, which was the reciprocal of the dilution. 2.4 Isolation of pure culture Discrete well-developed and separated colonies on the surface of a nutrient medium plate culture were each picked up with a sterile niddle and transferred separately in RCV medium slant. Each of these new slant cultures represents the growth of a single bacterial species. The colonies, which are different in appearances and characters were picked and purified. 2.5 Preparation for microscopic examination The strains were studied following the methods of Cerney (1993) including morphological and physiological features. Gram staining was used for the study of the bacterial morphology. Bacteria were grown on nutrient slants for overnight incubation at 30±0.5 ⁰C. A portion of the bacterial culture was taken out by a sterile loop and was suspended in sterile normal saline. The suspension was sufficiently diluted. A drop of suspension was taken on the slide and was spread evenly covering an area of about 15-20 mm diameter. The slide was then kept in a safe place to air-dry. The smear was fixed by rapidly passing the dry slide; smear uppermost three times through the flame of a Bunsen burner. After passing the slide through the flame, it should be possible to lay the slide on the back of the hand without the hand feeling uncomfortably hot. The slide was allowed to cool before staining. 2.6 Identification of gram stain A dried fixed smear was covered with oxalate crystal violet reagent for sixty seconds. The strain was then rapidly washed off with clean water. All the water was then tipped off and the smear was covered with Lugol’s iodine for sixty seconds. The iodine was then washed off with clean water. The smear was then decolorized rapidly for 10 seconds with acetone alcohol and was washed immediately with clear water. Finally, the smear was covered with safranine for 30 seconds. The slide was then washed thoroughly in water and blotted dry. The smear was examined under microscope by using high power (5 x 1000) immersion oil objective. The gram-negative organisms were stained pink and the gram positives were dark violet in color. 2.7 Physiological studies of the selected strains The physiological activities of the selected strains were tested through oxidase, catalase motility indole urease (MIU), methyl red (MR), aceton production (Voges-Proskauer), nitrate reduction, citrate utilization, hydrogen sulfide (H2S) production, gelation liquification and carbohydrate fermentation methods as demonstrated by Collee and Miles (1989).2.8 Maintenance of culture Stock cultures were maintained in soft agar (0.7% agar in nutrient broth, Difco Lab, Detroit) stab on one-dram airtight screw capped tubes, stored at 4 to 8⁰C. The working cultures were maintained on Trypticase Soyagar (TSA) slant in one dram airtight screw capped (150 x 16 mm) test tubes and stored at 4 to 8⁰C. Isolated colonies were then streaked on TSA slant in 5 mm screw capped test tubes and incubated overnight at 37±0.5⁰C. The TSA slant cultures were stored at 4 to 8⁰C and were used as working culture. For routine culture or routine use, culture was transferred from TSA slant on TSA plate.

Source of Information: Journal of the Faculty of Environmental Science and Technology. Okayama University Vol.13 No1, pp.97-101, March 2008

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Achievement/Findings:

The 34.6% of the total isolates was identified as nitrogen fixing microorganisms during the seedling (30 days after seed sowing) stage of rice (BR10) rhizosphere soil. Based on the selection criteria, the four individual strains were microbiologically identified. Their biochemical tests were strictly similar to Enterobacter spp., forstrain-1, Klebsiella spp. for strain-2, Bacillus spp. for strain-3 and Azospirillum spp. for strain-4. They were anaerobic in nature.

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